2008 Fall/Winter Paths of Progress

Journey to the center of cancer diagnosisBy Prashant Nair

Betsy Grant (right), who is taking an experimental drug to combat a brain tumor, enjoys the view with her mother, Barbie.

In August 2007, Betsy Grant, a 33-year-old marketing employee from Holliston, Mass., began to experience worrisome symptoms. She described some of them – nausea, moodiness, and menstrual disruptions – to her gynecologist, who suspected that Grant's hormone levels were awry, possibly due to a non-invasive and easily managed growth on her pituitary gland. A magnetic resonance imaging (MRI) scan revealed something far more serious – a brain tumor.

Neurosurgeons at Dana-Farber/Brigham and Women's Cancer Center (DF/BWCC) removed a lozenge-sized tumor from the right frontal lobe of Grant's brain, diagnosed the next week as a grade II oligoastrocytoma – a brain tumor with a tendency to recur. In the days that followed, scientists at Dana-Farber and Brigham and Women's Hospital (BWH) ran a series of molecular tests, some of which were developed by DF/BWCC neuropathologist Keith Ligon, MD, PhD, which showed that some of Grant's cells were positive for OLIG2 and CD68 – both proteins used for molecular cancer diagnosis.

Based on the clinical characteristics of Grant's tumor, DF/BWCC oncologist Andrew Norden, MD, suggested trying an experimental drug called vorinostat, in addition to a standard drug and radiation. Norden hopes that vorinostat, which has shown promise in treating some brain tumors, will "silence" the action of certain enzymes that prod Grant's tumor cells to grow unchecked. Grant says, "I'm happy to know that, if this medication gets approved, it would not only benefit me but other people, too."

A vital partnership

Different-colored chemical stains applied to thin slices of tissue show whether cells are normal or cancerous.

With such precisely targeted drugs, Dana-Farber and BWH researchers are inching closer to the promise of personalized medicine – tailoring cancer therapies to the genetic makeup of individual patients and their cancers. To be successful, personalized medicine requires collaboration between scientists and clinical investigators located in labs at Dana-Farber and BWH.

To aid in that collaboration, Edward J. Benz Jr., MD, president of Dana-Farber, and Michael Gimbrone Jr., MD, chairman of the pathology department at BWH, organized a task force on molecular pathology to explore personalized medicine for patients of DF/BWCC.

"Most people are not aware of the central role of pathology in categorizing tumors for treatment and in monitoring response to drugs – processes that cut to the heart of personalized cancer therapy," says DF/BWCC pathologist Massimo "Max" Loda, MD, who chairs the pathology task force with Janina Longtine, MD, co-director of the BWH Center for Advanced Molecular Diagnostics.

"The pathologist's report lends credence to the oncologist's treatment decision. It's a vital partnership, right from the get-go."– Massimo Loda, MD

"The pathologist's report lends credence to the oncologist's treatment decision," Loda adds. "It's a vital partnership, right from the get-go."

Pathology is a branch of medicine dedicated to the diagnosis of disease through the visual examination of cells, tissues, organs, and organ systems. Pathology services for DF/BWCC patients are provided through a lab at BWH. For research, Dana-Farber has opened a new Dana-Farber/Brigham and Women's Center for Molecular Oncologic Pathology (CMOP), directed by Loda; though located at Dana-Farber, it is a joint venture.

A tumor's journey

Samples from tumors removed by surgeons embark on a journey through diagnostic and research labs.

Patients at DF/BWCC begin a journey that may include surgery, chemotherapy, and radiation. But few know that a little piece of their cancer goes on its very own journey.

Spring 2008. It's late on a Friday afternoon when a team of neurosurgeons removes a small section of tan-pink tumor from the right frontal lobe of a patient's brain. Although the surgery is meticulously calculated, the surgeons depend on the expertise of the attending neuropathologist to ensure that the tissue they removed came from the tumor. They await the pathologist's diagnosis as the tissue is brought from the operating room into a small sideroom.

This "frozen section room" is abuzz with activity, like a command center of residents hunched over microscopes.Within minutes, a pathologist's assistant snap-freezes the tumor, cuts a thin slice, and mounts it on a slide. The slide is then dipped in a series of glass beakers with dyes, an arrangement not unlike an artist's palette. The dyes, hematoxylin and eosin, stain different components of the tumor cells to make them more visible for neuropathologist Rebecca Folkerth, MD.

Folkerth enters the room, glasses perched like a pince-nez, with her pathology resident in tow. She peers into the microscope, looking for signs of malignancy. Dense, blue-tinged blotches of tiny cell nuclei reveal telltale signs of runaway cell division. Blood vessels crisscross the tumor's crinkled landscape, indicating its greed for nutrients. Folkerth diagnoses the tumor as a likely high-grade astrocytoma – an aggressive, recurrent brain tumor. The pronouncement is preliminary, but it serves the surgeons' purpose; they remove the rest of the tumor, and the 58-year-old patient is later informed of the tentative diagnosis.

Pathology resident Shakti Ramkissoon of BWH places samples into containers to be preserved.

Before the tumor can continue its journey – to the histology room, to the CMOP at Dana-Farber, and to the molecular diagnostics lab at Brigham and Women's – Folkerth sets aside most or all of the tissue for overnight preparation in order to make an official diagnosis.

This process begins with Shakti Ramkissoon, MD, PhD, a pathology resident, who "fixes" the tumor by bathing it in formalin to ensure that its features remain intact. He then places bits in small plastic receptacles and loads them into a processing machine that dehydrates the tissue overnight.

Stained samples reveal to an experienced pathologist the typical abnormalities of cancer cells.

The next morning, technicians in the histology room slice and stain thin sections before Folkerth and a small group of residents and fellows gather to determine the type and severity of the tumor. Folkerth scans the sections, detecting abnormalities an untrained eye might miss. She notices a spike in cell density, a sign of frenetic cell division, clumps of calcium typical of certain brain tumors, flecks of red spotting that indicate thickened blood vessel walls, and branching thickets of blood vessels where there should be only a few, if any. Folkerth also detects signs of infiltration – tumor cells have diffused through the brain to wreak even more havoc.

A pathologist's work, Folkerth explains, is to describe in detail the characteristics of every sample, combine that description with knowledge, and decide what additional tests are needed to clinch the diagnosis. From this final pathology report, the oncologist discusses the findings and treatment options with the patient. Herein ends one leg of the tumor's journey through the pathologists' workshop.

A second part of the journey began earlier, back in the frozen section room. If enough tissue is available, a tiny portion will be saved for researchers at Dana- Farber, where the scientists examine the tissue for clues that might guide treatment.

The research side

The Center for Molecular Oncologic Pathology is a veritable jungle of high-tech instruments, extending the length of the second floor of Dana-Farber's Jimmy Fund Building. "Our goal is to marry clinical pathology, currently used to diagnose patients, with research pathology performed at CMOP. That marriage is likely to shape the future diagnosis and treatment of cancer," says Ligon, who pioneered the use of OLIG2 as a diagnostic indicator of brain tumors. Today, the test is a component of clinical diagnosis for many DF/BWCC patients.

Microscopes, array scanners, laser devices, and cutting instruments compete for space in bays occupied by technicians, postdoctoral fellows, and graduate students. Matthew Theisen, a technician in Ligon's lab, cuts the tiny, buff-colored sample from the operating room into fine bits, some of which he grows in a nutrient-rich slurry. This mixture prods the tumor stem cells – a small group of perpetually dividing cells purported to fuel the cancer – to grow into tiny spheres that can be studied in the lab two weeks later.

At that time, Theisen looks for molecular markers that define the tumor. These markers include EGFR, a growth-signaling protein implicated in many cancers; OLIG2, a marker for cancer stem cells; and KI67, a protein that reveals accelerated cell division. Seen through the microscope, these proteins look like magnified leopard skin, and reveal the tumor's aggressiveness. Theisen detects signs of rampant cell division and cues suggesting how that growth might be slowed or stopped.

Other clues come from more involved CMOP techniques, such as array CGH (comparative genomic hybridization), which singles out hyperactive and underperforming genes. Tissue microarrays can be used to simultaneously probe multiple samples for cancer-indicating proteins. Laser capture microdissection uses a focused laser beam to isolate suspicious parts of the tumor and air-lift them onto a receptacle for further tests.

Beyond diagnosis

CMOP scientists go beyond molecular diagnosis to help inform treatment options. For example, they are developing techniques that sabotage cancer genes to keep tumor growth in check. Ahmed Idbaih, MD, PhD, a postdoc in Ligon's lab, uses molecules, called small interfering RNAs (chemical cousins of DNA), to switch off the production of proteins, called transcription factors, which are thought to keep brain tumor stem cells from maturing into adult brain cells. Blocking these factors, Idbaih surmises, will slam the brakes on tumor growth in patients.

Extending the effort to use the data generated at the CMOP, Claire Sauvageot, PhD, a scientist in the laboratory of neurobiologist Charles Stiles, PhD, tests drugs from pharmaceutical companies on cells grown in labs and in mice.

Making use of leads from CMOP studies of brain tumors, Claire Sauvageot in Charles Stiles' lab is testing drugs targeted to molecular defects in glioblastoma.

"With glioblastoma, we know some of the molecular defects that cause the tumor cells to become malignant, so we test small-molecule inhibitors against them," Sauvageot says. Using the genetic clues gleaned from the analyses done by Ligon's group, Sauvageot determines whether the inhibitors might kill brain tumor cells of a certain genetic stripe. "That's the idea of targeted therapeutics. If we see something promising, we move it rapidly to clinical trials for gliomas," she says.

Besides going to CMOP, the sample also goes to the Molecular Diagnostics lab at BWH. Tests there pinpoint genetic defects in tumors, which might aid diagnosis and treatment decisions. At the lab, a technique called MGMT methylation analysis aims to determine which patients might benefit from the cancer drug Temodar®. Jesse Ladner, a wiry, soft-spoken technologist, analyzes the sample using a technique that distinguishes methylated DNA – which is attached to a chemical molecule known as a methyl group – from unmethylated DNA. Methylation of MGMT, a specific gene on the DNA, "silences" that gene, which might allow Temodar to kill tumor cells effectively.

"The pathologist is right at the center of personalized medicine, and the task force is aimed at making sure we're at the absolute cutting edge of expertise."– Dana-Farber President Edward J. Benz Jr., MD

Having provided a wealth of molecular information to guide treatment, the tumor's journey ends here. But the mission of the task force on molecular pathology has just begun.

"The pathologist is right at the center of personalized medicine, and the task force is aimed at making sure we're at the absolute cutting edge of expertise," says Dana-Farber President Benz. As it seeks to develop custom-designed treatments for cancer, the task force is already on its way to harnessing pathology's potential.